The role of polarity establishment components

A set of proteins that are critical for bud formation in yeast are the polarity establishment proteins. These include Cdc42p, a GTPase most closely related to members of the Rho family, and its GEF, Cdc24p (Adams et al., 1990; Sloat et al., 1981; Zheng et al., 1994). Cells containing temperature-sensitive mutations in either of these genes fail to form buds and form large, round, unbudded cells with multiple nuclei (Adams et al., 1990; Field and Schekman, 1980; Sloat and Pringle, 1978; Sloat et al., 1981). At restrictive temperature, these strains fail to localize many polarized components important for yeast budding properly, including Spa2p, actin patches, and septins (Adams and Pringle, 1984; Adams et al., 1990; Johnson and Pringle, 1990; Snyder et al., 1991; Ziman et al., 1991).

In localization studies Cdc42p was detected at polarized sites of growth, whereas Cdc24p, its GEF, localizes over the entire cell periphery (Pringle et al., 1995). Therefore, either Cdc24p functions only at polarized growth sites where Cdc42p accumulates or it has additional targets besides Cdc42p (Madden and Snyder, 1998). Published data from Zheng et al. (1993) suggest that Cdc24p regulates the activity of Cdc42p.

In mammalian cells, Cdc42p interacts with the PAK protein kinase to help mediating cell polarization (Manser et al., 1994; Martin et al., 1995). Yeast cells contain three PAK kinase homologs, Ste20p (see chapter 1.3.5.3), Cla4p, and Skm1p (Cvrcková et al., 1995;

Martin et al., 1997). Strains containing either ste20Δ or cla4Δ or skm1Δ are viable and do not exhibit any apparent defects (Cvrcková et al., 1995; Martin et al., 1997). Interestingly, ste20Δ cla4Δ double mutant strains are not viable indicating that the functions between these two

INTRODUCTION

19 kinases overlap (Cvrcková et al., 1995). Combinations between the skm1Δ mutation with either ste20Δ or cla4Δ produced no detectable phenotype indicating that Skm1p is not redundant with Ste20p or Cla4p (Martin et al., 1997), but the exact function of Skm1p is not known. Further studies revealed that Ste20p and Cla4p interact physically with Cdc42p and that this association is important for the function(s) of these proteins (Cvrcková et al., 1995, Leberer et al., 1997; Peter et al., 1996; Simon et al., 1995).

Another protein that helps to establish and maintain polarity in yeast is Bni1p. Bni1p is a member of the highly conserved formin protein family found in S. cerevisiae (Evangelista et al., 1997), S. pombe (Chang et al., 1997; Petersen et al., 1995), mouse (Torres et al., 1991), and Drosophila (Castrillon and Wasserman, 1994; Emmons et al., 1995). Bni1p associated with actin in two-hybrid assays and with regulators of the actin cytoskeleton (Cdc42p) and its effectors (Ste20p and Cla4p) in co-immunoprecipitation or in vitro binding experiments (Evangelista et al., 1997). Diploid bni1Δ mutant strains bud randomly both in the first division and in subsequent division (Zahner et al., 1996), but bni1Δ haploid cells bud normally. Thus, Bni1p might play an important role in the establishment of the distal tag in diploid daughter cells.

Other components that genetically interact with Cdc42p and Cdc24p have been identified. These include Bem3p, a Rho-GAP homolog that serves as a GTPase activating protein for Cdc42p in vitro (Stevenson et al., 1995; Zheng et al., 1993; Zheng et al., 1994).

Rga1p and Rga2p, two Rho-GAP homologs, serve as GAPs for Cdc42p in vivo (Stevenson et al., 1995). Moreover, both proteins are involved in control of septin organization, pheromone response, and haploid invasive growth (Smith et al., 2002). Zds1p and Zds2p appear to down-regulate Cdc42p in vivo (Bi and Pringle, 1996). Mutations in another polarity establishment protein, BEM1, are co-lethal with MSB1, a high-copy suppressor of both cdc24 and cdc42 (Bender and Pringle, 1991). Bem1p is an SH3-domain protein that physically interacts with Cdc24p, Ste5p, and Ste20p (Leeuw et al., 1995). It could be shown that this protein strongly facilitates bud emergence, possibly as a scaffold to assist the clustering of Cdc24p-Cdc42p (Pruyne and Bretscher, 2000a). Finally, two potential targets of Cdc42p, Gic1p and Gic2p, have been described recently; Gic1p and Gic2p interact genetically with Cdc42p and contain a CRIB domain, which is characteristic of many Cdc42p-interacting proteins (Brown et al., 1997; Chen et al., 1997). Bem1p, Gic1p, Gic2p, Zds1p, and Zds2p are all important for cell polarity in yeast, and each of these proteins except Zds2p is localized to sites of polarized cell

INTRODUCTION

growth, similar to Cdc42p (Bi and Pringle, 1996; Brown et al., 1997; Chen et al., 1997;

Pringle et al., 1995).

1.3.8 A model for choosing bud sites in the axial and bipolar pattern

Both haploid and diploid yeast cells use spatial cues for producing the axial or bipolar budding pattern. In case of haploid cells, Bud10p is assumed to function as marker protein for the axial budding process (Fig. 5). The extracellular domain of Bud10p is highly glycosylated and may serve to anchor the protein in the cell wall. Besides Bud10p, septins, Bud3p, and Bud4p are also part of the so-called cytokinesis tag, and the tight clustering between these proteins presumably helps to generate a potent signal (Roemer et al., 1996; Halme et al., 1996). Kang et al. (2001) could show that Bud10p directly interacts with Bud5p, which is a component of the Bud1p GTPase signaling module. Local activation of the Bud1p GTPase in turn activates the Cdc42p GTPase, which leads to recruitment of other proteins required for establishment of polarized growth.

Diploid yeast cells use spatial cues for producing the bipolar pattern that are entirely distinct from those used in the axial pattern (Chant, 1999). In diploids, Bud8p and Bud9p were proposed to function as bipolar landmarks at the distal and proximal pole, respectively (Fig. 5). As described before, sequence analyses on Bud8p and Bud9p predict related transmembrane proteins that are highly glycosylated (Harkins et al., 2000). A physical interaction between Bud8p and Bud5p was shown by Kang et al. (2004a), an association between Bud9p and Bud5p is demonstrated in this work. It is supposed that the same components needed for polarity establishment in haploids are involved in further signal transduction that finally leads to recruitment of components essential for polarized growth.

INTRODUCTION

21 Fig. 5: Molecular machinery guiding the axes of polarization during budding. Both haploid and diploid yeast cells use spatial cues for producing the axial and bipolar budding pattern, respectively. In haploid yeast cells, Bud10p functions as spatial landmark protein for axial budding. In diploids, Bud8p and Bud9p fulfill a function as distal and proximal pole marker, respectively. These markers interact with Bud5p, a component of the Bud1p GTPase signaling module. Local activation of the Bud1p GTPase in turn activates the Cdc42p GTPase, which leads to recruitment of other proteins required for establishment of polarized growth.

INTRODUCTION

1.4 Aim of this Work

Although genetic and cell biological analysis has led to the identification of a large number of components that constitute the bud site selection pathway in diploid yeast cells, the molecular function of the involved factors is poorly understood in many cases. Within the scope of this work, it was intended to better characterize individual components that play a role in the process of bud site selection in yeast.

The potential landmark proteins Bud8p and Bud9p are objects of peculiar interest. It is known that both Bud8p and Bud9p localize to the distal and the proximal pole, respectively, to fulfill their function as landmarks in the bipolar budding process of diploid cells. In former studies concerning the localization of Bud8p and Bud9p, the proteins were each investigated separately. To get a better insight in appearance of the landmark proteins, one purpose of this study should be the realization of co-localization experiments enabling the detection of both Bud8p and Bud9p within the same cell.

As described in above, the overall structure of Bud8p and Bud9p is similar in that both proteins are predicted to consist of a large NH2-terminal extracellular domain, followed by a membrane-spanning domain (TM1), a short cytoplasmic loop, a second membrane-spanning domain (TM2), and a very short extracellular domain at the COOH-terminus. The NH2 -terminal portion of both proteins contains several N- and O-glycosylation sites that appear to be functional. However, characterization of distinct domains of the landmark proteins that are required for, e.g., correct function of the proteins, transport of the proteins to the cell poles, or that confer interaction with other proteins or downstream components of the budding machinery are not known. To answer this these questions, a systemic analysis of Bud8p and Bud9p should be carried out to better understand the structure and the function of the bipolar landmark proteins. Furthermore, deletion sets resulting from systemic analysis of Bud8p and Bud9p should be used to investigate the existing association between Bud8p and Bud9p and their interaction partners Bud5p and Rax1p via co-immunoprecipitation experiments.

In a last approach, investigations should be carried out to get more information on the intracellular transport of Bud8p and Bud9p to the yeast cell poles. Therefore, tagged versions of both proteins should be investigated by 'pulse-chase' experiments, which should allow predictions about secretion of the proteins via the secretory pathway and their half-life periods within the cell.

23

2. Materials and Methods

2.1 Materials

2.1.1 Chemicals, enzymes, and antibodies

Chemicals used for the production of solutions, buffers and culture media were sourced from MERCK (Darmstadt, D), ROCHE GMBH (Mannheim, D), CARL ROTH GMBH & CO KG (Karlsruhe, D), INVITROGEN GMBH (Karlsruhe, D), FLUKA (Neu-Ulm, D) and SIGMA -ALDRICH CHEMIE GMBH (Steinheim, D).

Restriction enzymes, DNA modifying enzymes as well as Taq and Pfu polymerase were provided by NEW ENGLAND BIOLABS (Ipswich, MA, USA), MBI FERMENTAS (Vilnius, Lit) and PROMEGA (Madison, WI, USA). KOD HIFI DNA polymerase is fabricated by NOVAGEN (Darmstadt, D). As DNA size standards 'GENE RULER 1 kb DNA ladder Plus' from MBI FERMENTAS (Vilnius, Lit) and '1 kb DNA ladder' from NEW ENGLAND BIOLABS

(Ipswich, MA, USA) were used. Agarose for preparation of gels was provided from CARL

ROTH GMBH & CO KG. Preparation of plasmid DNA from Escherichia coli and extraction of DNA from gels was carried out using kits from QIAGEN (Hilden, D).

DNA sequencing chemicals were provided by APPLIED BIOSYSTEMS GMBH (Weiterstadt, D). Synthetic oligonucleotides were purchased from INVITROGEN. Bradford solution for the determination of protein contents was provided by BIORAD INDUSTRIES GMBH (München, D).

'See Blue Pre-Stained' (Novex, San Diego CA, USA), 'Prestained Protein Molecular Weight Marker' (MBI FERMENTAS) as well as 'Rainbow marker RPN 756' from AMERSHAM

LIFE SCIENCE (Uppsala, S) were used as marker for determination of protein weight. SDS protein gels were blotted on nitrocellulose membrane obtained from SCHLEICHER & SCHUELL

(Dassel, D). Antibodies were produced by MOLECULAR PROBES (Eugene, OR, USA) and SANTA CRUZ BIOTECH INC. (Santa Cruz, CA, USA) and detected on Hyperfilm™-ECL™

(AMERSHAM PHARMACIA BIOTECH, Buckinghamshire, GB).

2.1.2 Yeast strains, plasmids, and oligonucleotides

The yeast strains used in this study are listed in Table 1. All strains are congenic to the Σ1278b genetic background. The construction of corresponding strains containing mutant alleles of BUD8 and BUD9, respectively, and/or tagged genes is described below. Standard

MATERIALS AND METHODS

methods for transformation and genetic crosses were used, and standard yeast culture YPD, YNB and SC media were prepared essentially as described (Guthrie and Fink, 1991).

Plasmids used in this study are listed in Table 2, their construction is described below;

sequences of oligonucleotides used in this study are listed in Table 3.

Table 1: Saccaromyces cerevisiae strains used in this study

Strain Genotype Source

RH2448 a/α rsr1Δ::kanR/rsrΔ1::kanR ura3-52/ura3-52 leu2::hisG/LEU2 trp1::hisG/TRP1

Taheri et al., 2000

RH2449 a/α bud8Δ::HIS3/bud8Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

Taheri et al., 2000

RH2453 a/α bud8Δ::HIS3/bud8Δ::HIS3 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

Taheri et al., 2000

RH2495 a/α ura3-52/ura3-52 leu2::hisG/leu2::hisG his3::hisG/HIS3 trp1::hisG/TRP1

Taheri et al., 2000

RH2905 a myc⁹-BUD9-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2906 a myc⁹-BUD9^Δ8-48-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2907 a myc⁹-BUD9^Δ8-130-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2908 a myc⁹-BUD9^Δ91-130-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2909 a myc⁹-BUD9^Δ91-218-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2910 a myc⁹-BUD9^Δ168-218-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2911 a myc⁹-BUD9^Δ168-283-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2912 a myc⁹-BUD9^Δ244-283-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2913 a myc⁹-BUD9^Δ244-369-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2914 a myc⁹-BUD9^Δ323-369-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2915 a myc⁹-BUD9^Δ323-450-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2916 a myc⁹-BUD9^Δ406-450-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2917 a myc⁹-BUD9^Δ406-544-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

MATERIALS AND METHODS

25

RH2918 a myc⁹-BUD9^Δ460-544-TRP1 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2919 α myc⁹-BUD9-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2920 α myc⁹-BUD9^Δ8-48-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2921 α myc⁹-BUD9^Δ8-130-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2922 α myc⁹-BUD9^Δ91-130-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2923 α myc⁹-BUD9^Δ91-218-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2924 α myc⁹-BUD9^Δ168-218-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2925 α myc⁹-BUD9^Δ168-283-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2926 α myc⁹-BUD9^Δ244-283-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2927 α myc⁹-BUD9^Δ244-369-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2928 α myc⁹-BUD9^Δ323-369-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2929 α myc⁹-BUD9^Δ323-450-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2930 α myc⁹-BUD9^Δ406-450-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2931 α myc⁹-BUD9^Δ406-544-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

RH2932 α myc⁹-BUD9^Δ460-544-LEU2 bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG

this study

YHUM829 a/α ura3-52/ura3-52 his3::hisG/his3::hisG leu2::hisG/leu2::hisG strain collection H.-U. Mösch YHUM992 a/α ^bud8Δ::HIS3/bud8Δ::HIS3 his3::hisG/his3::hisG leu2::hisG/LEU2

trp1::hisG/TRP1

this study

YHUM993 a/α bud9Δ::HIS3/bud9Δ::HIS3 his3::hisG/his3::hisG ura3-52/ura3-52 TRP1/trp1::hisG leu2::hisG/LEU2

this study

YHUM904 a bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG strain collection H.-U. Mösch YHUM861 α bud8Δ::HIS3 ura3-52 his3::hisG leu2::hisG strain collection

H.-U. Mösch YHUM994 a bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG this study YHUM995 α ^bud9Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG this study

MATERIALS AND METHODS

YHUM842 a/α ^myc6-BUD8-URA3/myc⁶-BUD8-URA3 bud8Δ::HIS3/bud8Δ^::HIS3 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

YHUM865 a myc⁶-BUD8-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM866 a myc⁶-BUD8^Δ7-53-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM867 a myc⁶-BUD8^Δ7-114-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM870 a myc⁶-BUD8^Δ74-114-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM871 a myc⁶-BUD8^Δ74-216-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study

MATERIALS AND METHODS

27

YHUM872 a myc⁶-BUD8^Δ173-216-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM873 a myc⁶-BUD8^Δ173-325-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM874 a myc⁶-BUD8^Δ268-325-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM875 a myc⁶-BUD8^Δ268-417-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM876 a myc⁶-BUD8^Δ375-417-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM877 a myc⁶-BUD8^Δ375-505-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM878 a myc⁶-BUD8^Δ468-505-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM879 a myc⁶-BUD8^Δ513-600-URA3 bud8Δ::HIS3 ura3-52 his3::hisG trp1::hisG this study YHUM882 α ^myc6-BUD8-URA3 bud8Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM883 α ^{myc6-BUD8Δ7-53-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM884 α ^{myc6-BUD8Δ7-114-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM887 α ^{myc6-BUD8Δ74-114-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM888 α ^{myc6-BUD8Δ74-216-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM889 α ^{myc6-BUD8Δ173-216-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM890 α ^{myc6-BUD8Δ173-325-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM891 α ^{myc6-BUD8Δ268-325-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM892 α ^{myc6-BUD8Δ268-417-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM893 α ^{myc6-BUD8Δ375-417-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM894 α ^{myc6-BUD8Δ375-505-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM895 α ^{myc6-BUD8Δ468-505-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study YHUM896 α ^{myc6-BUD8Δ513-600-URA3 bud8}Δ::HIS3 ura3-52 his3::hisG leu2::hisG this study

YHUM1007 a bud8Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG strain collection H.-U. Mösch YHUM1008 α bud8Δ::HIS3 ura3-52 his3::hisG leu2::hisG trp1::hisG strain collection

H.-U. Mösch YHUM1009 a/α ^myc9-BUD9-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9-LEU2

bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1010 a/α myc⁹-BUD9^Δ8-47-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ8-47-LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1011 a/α ^{myc9-BUD9Δ8-130}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ8-130-LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1012 a/α myc⁹-BUD9^Δ91-130-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ91-130-LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1013 a/α ^{myc9-BUD9Δ91-218}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ91-218-LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1014 a/α myc⁹-BUD9^Δ168-218-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ168-218 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1015 a/α ^{myc9-BUD9Δ168-283}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ168-283 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1016 a/α myc⁹-BUD9^Δ244-283-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ244-283 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

MATERIALS AND METHODS

YHUM1017 a/α ^{myc9-BUD9Δ244-369}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ244-369 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1018 a/α myc⁹-BUD9^Δ323-369-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ323-369 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1019 a/α ^{myc9-BUD9Δ323-450}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ323-450 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1020 a/α myc⁹-BUD9^Δ406-450-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ406-450 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1021 a/α ^{myc9-BUD9Δ406-544}-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ406-544 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1022 a/α myc⁹-BUD9^Δ460-544-TRP1/trp1::hisG leu2::hisG/myc⁹-BUD9^Δ460-544 -LEU2 bud9Δ::HIS3/bud9Δ::HIS3 ura3-52/ura3-52 his3::hisG/his3::hisG

this study

YHUM1023 a/α ^bud8Δ::HIS3/BUD8 myc⁶-BUD8-URA3/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

this study

YHUM1024 a/α bud8Δ::HIS3/BUD8 URA3/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

this study

YHUM1025 a/α ^bud8Δ::HIS3/BUD8 myc⁶-BUD8^Δ173-216-URA3/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

YHUM1025 a/α ^bud8Δ::HIS3/BUD8 myc⁶-BUD8^Δ173-216-URA3/ura3-52 his3::hisG/his3::hisG leu2::hisG/LEU2 trp1::hisG/TRP1

Im Dokument Structure-Function Analysis of the Cell Polarity Determinants Bud8p and Bud9p in <i>Saccharomyces cerevisiae</i> (Seite 26-0)